Bottom Line:
The following observations were made: (a) T cell hybridomas derived from tolerance-broken animals required higher concentrations of MCC 88-103 to be stimulated than hybridomas derived from normal immune animals, suggesting that they have T cell receptors (TCRs) of lower affinity; (b) in contrast to normal immune animals whose MCC-specific TCRs are typically Vbeta3(+)/Valpha11(+), none of the hybridomas derived from tolerance-broken animals expressed Vbeta3, although they were all Valpha11(+).Also, the Vbeta complementarity determining region 3 (CDR3) regions from the tolerance-broken animals did not contain the canonical structure and length characteristics of the normal MCC 88-103 immune repertoire; and (c) adoptive transfer and tolerization of MCC-specific Vbeta3(+)/Valpha11(+) transgenic T cells followed by immunization with heteroclitic antigen failed to terminate the state of tolerance.Collectively, these data strongly suggest that the mechanism involved in breaking tolerance in this system is the stimulation of nontolerized, low-affinity clones, rather than reversal of anergy.

ABSTRACTH-2K mice injected, intravenously in saline or intraperitoneally in incomplete Freund's adjuvant, with large quantities of the immunodominant I-E(k)-restricted epitope from moth cytochrome c (MCC) 88-103 fail to respond to subsequent immunization with this epitope when administered in complete Freund's adjuvant. This state of tolerance can be broken by immunization with certain MCC 88-103 analogues that are heteroclitic antigens as assessed on representative MCC 88-103 specific T cell clones. In this paper, the mechanism of breaking tolerance by heteroclitic antigens was investigated. The following observations were made: (a) T cell hybridomas derived from tolerance-broken animals required higher concentrations of MCC 88-103 to be stimulated than hybridomas derived from normal immune animals, suggesting that they have T cell receptors (TCRs) of lower affinity; (b) in contrast to normal immune animals whose MCC-specific TCRs are typically Vbeta3(+)/Valpha11(+), none of the hybridomas derived from tolerance-broken animals expressed Vbeta3, although they were all Valpha11(+). Also, the Vbeta complementarity determining region 3 (CDR3) regions from the tolerance-broken animals did not contain the canonical structure and length characteristics of the normal MCC 88-103 immune repertoire; and (c) adoptive transfer and tolerization of MCC-specific Vbeta3(+)/Valpha11(+) transgenic T cells followed by immunization with heteroclitic antigen failed to terminate the state of tolerance. Collectively, these data strongly suggest that the mechanism involved in breaking tolerance in this system is the stimulation of nontolerized, low-affinity clones, rather than reversal of anergy. Further support for this mechanism was the finding that after activation, T cells apparently have a lowered threshold with respect to the affinity of interaction with antigen required for stimulation.

Figure 2: Function and receptor analysis of T cell hybridomas derived from normal immune and tolerance-broken animals. (A) T cell hybridomas derived from mice which were pMCC (▴) or pMCC-A (▪) immunized, or tolerized to pMCC and immunized with pMCC-A (♦) were analyzed for their capacity to make IL-2 in response to varying doses of pMCC. (B and C) T cell hybridomas from pMCC-immunized mice (light line), pMCC-A–immunized mice (dotted line), and tolerance-broken mice (bold line) were analyzed for expression of CD3 (B) and CD4 (C). Dashed line indicates negative control staining.

Mentions:
The results shown in Fig. 1 and those reported previously 26 suggest that the T cells involved in the breaking of tolerance have a lower avidity for antigen compared with normal immune T cells, as evidenced by the shift in the antigen dose–response profile. To study in greater detail the functional activity of the T cells involved in the breaking of tolerance to pMCC, and in order to analyze the structure of their TCRs, a series of T cell hybridomas was made from animals whose tolerance to pMCC was broken by immunization with pMCC-A. As controls, hybridomas were also derived from normal animals immunized with either pMCC or pMCC-A. The response of representative hybridomas derived from normal or tolerance-broken animals to stimulation with pMCC is shown in Fig. 2. The two hybridomas derived from pMCC or pMCC-A normal immune animals behaved similarly to stimulation with pMCC. In contrast, and similar to the data obtained with the bulk T cell response, the hybridoma derived from the tolerance-broken group required ∼10-fold more pMCC to give an IL-2 response comparable to that of the hybridomas derived from the normal immunized animals. The decreased antigen responsiveness of the hybridomas derived from tolerance-broken animals was not due to a decrease in surface expression of either the TCR or the CD4 coreceptor (Fig. 2B and Fig. C; and data not shown).

Figure 2: Function and receptor analysis of T cell hybridomas derived from normal immune and tolerance-broken animals. (A) T cell hybridomas derived from mice which were pMCC (▴) or pMCC-A (▪) immunized, or tolerized to pMCC and immunized with pMCC-A (♦) were analyzed for their capacity to make IL-2 in response to varying doses of pMCC. (B and C) T cell hybridomas from pMCC-immunized mice (light line), pMCC-A–immunized mice (dotted line), and tolerance-broken mice (bold line) were analyzed for expression of CD3 (B) and CD4 (C). Dashed line indicates negative control staining.

Mentions:
The results shown in Fig. 1 and those reported previously 26 suggest that the T cells involved in the breaking of tolerance have a lower avidity for antigen compared with normal immune T cells, as evidenced by the shift in the antigen dose–response profile. To study in greater detail the functional activity of the T cells involved in the breaking of tolerance to pMCC, and in order to analyze the structure of their TCRs, a series of T cell hybridomas was made from animals whose tolerance to pMCC was broken by immunization with pMCC-A. As controls, hybridomas were also derived from normal animals immunized with either pMCC or pMCC-A. The response of representative hybridomas derived from normal or tolerance-broken animals to stimulation with pMCC is shown in Fig. 2. The two hybridomas derived from pMCC or pMCC-A normal immune animals behaved similarly to stimulation with pMCC. In contrast, and similar to the data obtained with the bulk T cell response, the hybridoma derived from the tolerance-broken group required ∼10-fold more pMCC to give an IL-2 response comparable to that of the hybridomas derived from the normal immunized animals. The decreased antigen responsiveness of the hybridomas derived from tolerance-broken animals was not due to a decrease in surface expression of either the TCR or the CD4 coreceptor (Fig. 2B and Fig. C; and data not shown).

Bottom Line:
The following observations were made: (a) T cell hybridomas derived from tolerance-broken animals required higher concentrations of MCC 88-103 to be stimulated than hybridomas derived from normal immune animals, suggesting that they have T cell receptors (TCRs) of lower affinity; (b) in contrast to normal immune animals whose MCC-specific TCRs are typically Vbeta3(+)/Valpha11(+), none of the hybridomas derived from tolerance-broken animals expressed Vbeta3, although they were all Valpha11(+).Also, the Vbeta complementarity determining region 3 (CDR3) regions from the tolerance-broken animals did not contain the canonical structure and length characteristics of the normal MCC 88-103 immune repertoire; and (c) adoptive transfer and tolerization of MCC-specific Vbeta3(+)/Valpha11(+) transgenic T cells followed by immunization with heteroclitic antigen failed to terminate the state of tolerance.Collectively, these data strongly suggest that the mechanism involved in breaking tolerance in this system is the stimulation of nontolerized, low-affinity clones, rather than reversal of anergy.

ABSTRACTH-2K mice injected, intravenously in saline or intraperitoneally in incomplete Freund's adjuvant, with large quantities of the immunodominant I-E(k)-restricted epitope from moth cytochrome c (MCC) 88-103 fail to respond to subsequent immunization with this epitope when administered in complete Freund's adjuvant. This state of tolerance can be broken by immunization with certain MCC 88-103 analogues that are heteroclitic antigens as assessed on representative MCC 88-103 specific T cell clones. In this paper, the mechanism of breaking tolerance by heteroclitic antigens was investigated. The following observations were made: (a) T cell hybridomas derived from tolerance-broken animals required higher concentrations of MCC 88-103 to be stimulated than hybridomas derived from normal immune animals, suggesting that they have T cell receptors (TCRs) of lower affinity; (b) in contrast to normal immune animals whose MCC-specific TCRs are typically Vbeta3(+)/Valpha11(+), none of the hybridomas derived from tolerance-broken animals expressed Vbeta3, although they were all Valpha11(+). Also, the Vbeta complementarity determining region 3 (CDR3) regions from the tolerance-broken animals did not contain the canonical structure and length characteristics of the normal MCC 88-103 immune repertoire; and (c) adoptive transfer and tolerization of MCC-specific Vbeta3(+)/Valpha11(+) transgenic T cells followed by immunization with heteroclitic antigen failed to terminate the state of tolerance. Collectively, these data strongly suggest that the mechanism involved in breaking tolerance in this system is the stimulation of nontolerized, low-affinity clones, rather than reversal of anergy. Further support for this mechanism was the finding that after activation, T cells apparently have a lowered threshold with respect to the affinity of interaction with antigen required for stimulation.